Post-translational conjugation of a protein by another protein, polysaccharide, lipid and nucleic acid, or any combination of the above plays a key role virtually in every aspect of cellular functions. A conjugated molecule is either a monomeric single-molecule or a polymeric macromolecule with either a linear or a branched structure. Macromolecules include, but are not limited to polysaccharides, adenosine diphosphate (ADP)-ribosyls, fatty acids, polynucleotides, glycosylphosphatidylinositol (GPI) anchors, proteins or polypeptides, ubiquitin, small ubiquitin-like modifier (SUMO), neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), interferon-stimulated gene 15 kDa (ISG15), and other ubiquitin-like molecules (UBLs).
Many types of human diseases display abnormal molecular conjugation. For example, abnormal glycosylation occurs in many types of cancers (Mehta and Block, 2008). Ubiquitin-containing conjugates are present in neurodegenerative diseases (Dohm et al., 2008). Telomeric aggregates accumulate in tumor cells (Mai and Garini, 2006). Advanced glycation adducts are found in samples obtained from patients with heart disease and diabetes (Thornalley, 2002; Meerwaldt et al., 2008). Disease-specific macromolecule-to-macromolecule conjugates are present in body fluids such as blood serum or cerebrospinal fluid (CSF), but few reliable methods are currently available to detect a macromolecule-to-macromolecule conjugate in situ or ex situ in a conjugation site-specific manner. Because macromolecule-to-macromolecule conjugation sites are either poorly antigenic or hidden antigens, antibodies to macromolecule-to-macromolecule conjugation sites are extremely difficult to make.
Methods of making antibodies against post-translational modified proteins in the form of a small monomeric molecule, including phosphorylation, acetylation, methylation, and nitrolization, are well established. In comparison to monomeric modification site-specific antibodies, there is no effective method currently available for making polymeric macromolecular conjugation site-specific antibodies. Macromolecular conjugation can be defined as covalent conjugation between two polymeric biomolecules, including, but not limited to, protein glycosylation, lipidation, ADP-ribosylation, ubiquitination, sumoylation, NEDDylation, ISGylation, GPI-anchor, transglutaminase-mediated cross-links, and the like.
In the post-genomic era, our knowledge of macromolecule-to-macromolecule conjugation and its relation to diseases has grown exponentially. For that reason, investigators have devoted extensive efforts to generation of macromolecule-to-macromolecule conjugation site-specific antibodies by conventional antigen design, antibody-making, and antigen detection methods. However, these efforts have been so far proven futile (Matsumoto et al., 2008). Therefore, there is an unmet need of polymeric conjugation site-specific antibodies because they have significant value.
Protein ubiquitination involves many biological processes. There are previous reports of generation of anti-polyubiquitin antibodies. Pirim (1998) reported an anti-polyubiquitin antibody. However, this antibody does not recognize isopeptide bond-branched ubiquitin-to-ubiquitin conjugation, which are dominant forms of cellular ubiquitin conjugates. Rather this antibody recognizes head-to-tail (c- to n-terminal conjugation) poly-ubiquitins.
Fujimuro et al. (2005) reported anti-polyubiquitin monoclonal antibodies named as FK1 and FK2. Both FK1 and FK2 antibodies recognize the polyubiquitin chain. However, there are two fundamental differences between making FK1 and FK2 antibodies, and the antibodies and methods described in the present invention: (i) FK1 and FK2 were made by using regular polyubiquitin antigens; and (ii) FK1 and FK2 cannot recognize the conjugation sites of ubiquitinated proteins (Fujimuro et al., 2005). Therefore, FK1 and FK2 are not conjugation site-specific antibodies, rather than general polyubiquitin antibodies.
There are reports and patents about methods and antibodies to the diglycine-linked lysine structure for profiling of ubiquitinated proteins with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Peng et al., 2003; Denis et al., 2007; Xu et al., 2010; Kim et al., 2011; U.S. Pat. No. 9,181,326). These antibodies against the diglycine-linked lysine structure were made either with the reaction products between lysine-rich histone III-S protein and t-butyloxycarbonyl-Gly-Gly-N-hydroxysuccinimide (Boc-Gly-Gly-NHS) (Xu et al., 2010), or using the synthetic diglycine-linked peptide library immunogen (U.S. Pat. No. 9,181,326). These antibodies were not produced for detecting hidden antigen in a sample in conventional antibody-based applications such as enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, and multiplex assay; or combinations thereof, but rather they were developed for the LC-MS/MS profiling of ubiquitinated peptides (Peng et al., 2003; Denis et al., 2007; Xu et al., 2010). In comparison, the inventive ACE methods and antibodies are produced for detecting hidden antigens in a sample in the above-mentioned conventional antibody-based applications. The inventive ACE methods and antibodies do not include those for capturing the diglycine-linked lysine structure in the LC-MS/MS profiling applications.
Matsumoto et al. (2008) generated two linkage-specific antibodies that recognize polyubiquitin chains through lysine 63 (K63) or 48 (K48) linkage (US patent 20070218069A) for the conventional antibody based applications. However, there are several fundamental differences between the method of making these two linkage-specific antibodies and the methods of the present invention. The “antibodies” made by Matsumoto et al. (2008) were not generated by conventional animal immunization methods, rather by a phage display approach of random screening of the ubiquitin conjugation site binders. This phage display approach has advantage to be able to select binding partners from millions of other irrelevant proteins, but these binding partners are “antibody-like” molecules. Also, the phage display approach usually has technical challenges associated with it. For instance, it is acknowledged that the affinity and specificity of binding partners generated by phage display may be suboptimal, relative to conventional immunoglobulin or antibody, and the loss of the original heavy- and light-chain pairings is also a challenge. Perhaps for these reasons, phage display has not been widely used to make “antibodies” reagents (Ward, 2002). In comparison, the present invention uses the Artificially Cleaved Epitope (ACE, see below) strategy for designing and detecting hidden macromolecular conjugation site-specific and linear antigens, which are proven to be effective and reliable.
There are several patented methods for making peptide antibodies. Patent WO 02/25287 describes methods for analysis of proteins by producing a mixture of peptides and contacting the mixture of peptides to filtering agents or antibodies in order to decrease the complexity of a mixture prior to the application of an analytical technique such as mass spectrometry. U.S. Pat. No. 7,460,960 described methods by the use of capture agents or antibodies that interact with the Proteome Epitope Tags (PETs) in a sample. However, these methods cannot be used to design and detect hidden antigens, and they are also principally and profoundly different with the methods of the present invention.
Currently, there are several cleavage site-specific antibodies commercially available. U.S. Pat. No. 7,803,553 described an antibody for detecting an active form of TGF-β1 naturally cleaved in vivo. U.S. Pat. No. 6,762,045 described an antibody to naturally cleaved caspase-3. All currently available cleavage-specific antibodies were developed to detect the naturally occurring cleavage sites in vivo, and cannot be used to detect hidden antigens such as macromolecule-to-macromolecule conjugation sites. In contrast, the present inventive methods are to design and detect “Artificially Cleaved Epitopes (ACEs, see below)” of hidden antigens that are not naturally present or exposed. The inventive ACE methods do not include those for detecting naturally cleaved epitopes in a sample.
Macromolecules other than polypeptides can also be used to generate antibodies successfully, including, but not limited to, antibodies to lipids, nucleic acids, and saccharide. For example, a mouse monoclonal antibody (e.g., CTD110.6) recognizing the single O-linked N-acetylglucosamine (GlcNAc) is commercially available. A mouse antibody (e.g., clone 26-5) to a lipid structure is also reported (Young et al., 1987). However, the common polysaccharide-to-protein and lipid-to-protein conjugation site-specific antibodies described in the present invention are not currently available, most probably because they are hidden antigens which cannot be detected by most conventional antibody-based methods.